Many proteolytic enzymes along with their cofactors and inhibitors play an important role in the proper functioning of the human body. These enzymes, which catalyze the cleavage of proteins, regulate many delicate and important biochemical processes. The clotting of blood represents a complex mechanism of regulated reactions. The essential process of blood coagulation is the proteolytic conversion of soluble fibrinogen into insoluble fibrin by the enzyme thrombin. Thrombin arises from its inactive precursor prothrombin through a series of proteolytic reactions involving protein, lipoprotein and inorganic ion cofactors. Inhibitors regulate blood clotting at various steps throughout the complex process.
The amounts of these enzymes contained in the body fluids lie within ranges considered "normal." However, the concentrations or activities of these enzymes, their cofactors or inhibitors, on occasion may either exceed or fall below their usual levels. Detecting the abnormal levels of these constituents of the body fluids assumes importance for, inter alia, the following two reasons.
Firstly, imbalance in the level of a particular enzyme, cofactor or inhibitor may directly and adversely affect the chemical reaction it regulates. Thus, in the case of blood clotting, thrombin promotes the formation of the clot. An insufficient level of thrombin results in inadequate blood clotting activity. Elevated thrombin can induce the formation of undesired clots with deleterious consequences. The reciprocal situation exists with antithrombin III levels, the inhibitor of thrombin. Elevated levels retard the clotting process while reduced levels may allow excessive clotting to occur. Plasmin, the primary protease of fibrinolysis, prevents the accumulation of fibrin clots. Excess levels, again, interfere with clotting while an insufficient level may permit the accumulation of undesired clots.
Secondly, such enzymes arise from biochemical reactions necessary for the maintenance of normal health. The concentration and activity of the enzymes is an indication as to the rate of these entire reactions themselves. Thus, detecting the levels of these enzymes can provide an indirect indication of an improper physical condition.
Several obstacles must be resolved before the detection of proteolytic enzymes can be used as a reliable diagnostic tool. Since these compounds exist in extremely small concentrations within the body fluids containing them, the usual chemical procedures are not sensitive enough to detect these enzymes.
An important advance in the quantitative determination of enzymes appears in U.S. Pat. No. 3,862,011 issued to R. E. Smith which discloses peptide derivatives of 4-methoxy-2-napthylamine. These compounds serve as substrates for proteolytic enzymes in tissue homogenates. The enzymes remove the 4-methoxy-2-napthylamine group from the peptide substrate. The cleaved group then reacts with a diazonium salt to form an azo dye. The intense color of the azo dye allows a spectrophotometric determination of concentration. The amount of the enzyme present is determined by comparison with a standard curve obtained by running similar tests with known enzyme concentrations.
U.S. Pat. No. 3,884,896 to G. E. B. Blomback et al. approaches the measurement of proteases in a manner similar to that described in the Smith patent. However, the Blomback et al. patent utilizes a chromophore, p-nitroaniline, attached to peptide molecules. The enzyme cleaves the chromophore which then undergoes direct spectrophotometric determination.